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Abstract:

The invention relates to method for increasing the yield of a plant grown
under conditions that cause periodic oxidative stress in said plant,
comprising the step of growing a seedling, tissue culture or plantlet
into a plant, and further comprising the step of manipulating said plant
or the environment of said plant such that an increase in the rate of
respiration and/or protein turnover in said plant due to said periodic
oxidative stress is intentionally prevented during the early ontogeny of
said plant.

Claims:

1. A method for increasing the yield of a plant grown under conditions
that cause periodic oxidative stress in said plant, comprising the step
of growing a seedling, tissue culture or plantlet into a plant, and
further comprising the step of manipulating said plant or the environment
of said plant such that said periodic oxidative stress is intentionally
prevented during the early ontogeny of said plant to maintain the AOX
component of the total dark respiration in said plant at levels
essentially equal to that of a plant that has never been exposed to
oxidative stress.

3. Method according to claim 1, wherein said AOX component of the total
dark respiration in said plant is close to zero.

4. Method according to claim 1, wherein said oxidative stress is
intentionally prevented by controlling the content of a reactive oxygen
species in the growth environment.

5. Method according to claim 1, wherein the periodic oxidative stress
during the early ontogeny of said plant is prevented by exposing the
plants to a level of CO2 that reduces the rate of respiration.

6. Method according to claim 4, wherein said reactive oxygen species in
the growth environment is O3 in the growth atmosphere.

7. Method according to claim 3, wherein said O3 exposure is
controlled by using O3 scrubbers and/or NOx scrubbers.

8. Method according to claim 3, wherein said O3 exposure is
controlled by exposing said plant or its growth atmosphere to exogenously
or endogenously produced isoprene.

9. Method according to claim 8, wherein said isoprene is exogenously
produced by optionally transgenic or recombinant microorganisms capable
of emitting isoprene.

10. Method according to claim 1, wherein the early ontogeny of said plant
is the prefloral stage or the vegetative stage, preferably a period from
1-6 months post-germination.

12. Method according to claim 1, wherein said method further comprises
the step of discontinuing the manipulation of said plant or the
environment of said plant when the ontogenic phase of said plant has
ended.

13. Method according to claim 1, wherein said method further comprises
determining prior to or simultaneously to growing said seedling, tissue
culture or plantlet into a plant: the total dark respiration and/or
respiration via the alternative oxidase (AOX) pathway in a plant of the
same variety; the length of the ontogenic phase in a plant of the same
variety; and/or the length and interval of the periodic conditions that
cause oxidative stress; and using said information in order to
intentionally prevent exposure of said plant to said periodic oxidative
stress during the early ontogeny of said plant, but not during the
post-ontogenic phase.

14. Method according to claim 13, wherein said information is used to
select the sowing or (trans)planting date for said plant in order to
essentially prevent exposure of said plant to said periodic oxidative
stress during the early ontogeny of said plant.

15. Method according to claim 1, wherein said the early ontogeny of said
plant is the vegetative or prefloral phase, and wherein the
post-ontogenic phase is the generative phase.

16. Method for increasing the average yearly crop production of a crop
production area, comprising growing a plant according to claim 1 and
using said plant as a crop plant in said production area.

17. Method according to claim 16, wherein said increase in the average
yearly crop production is brought about by an improved net carbon
fixation efficiency, biomass production, dry matter content and/or pest
resistance of said crop plant.

18. A plant obtainable by the method according to any claim 1, said plant
exhibiting a baseline respiration or a respiration via the alternative
oxidase (AOX) pathway that is significantly less than a plant of the same
variety grown under the same periodic conditions that cause oxidative
stress but not grown by the method of claim 1, wherein said plant is an
adult plant, and wherein said plant exhibits a first rate of respiration
under ambient ozone that is essentially equal to the rate of dark
respiration exhibited by said plant during early ontogeny, and wherein
said plant upon temporary exposure to ambient plus 100 ppbvof ozone
exhibits a second rate of respiration, which second rate is a significant
increase relative to said first rate, and wherein following said
temporary exposure said second rate of respiration returns to
pre-exposure levels.

19. Plant according claim 18, wherein said early ontogeny is the period
between germination and 0.5-6 months post germination.

20. Plant according to claim 18, wherein said early leaf ontogeny is the
period between emergence and 15-60 days after emergence of the first
leaves.

21. Plant according to claim 18, wherein the plant is a non-isoprene
emitter or a low-isoprene emitter.

23. Plant according to claim 18, wherein said plant exhibits a rate of
respiration via the alternative oxidase (AOX) pathway that is below 30,
preferably below 28, 27, 26 or 25% of the total dark respiration.

24. A plant part obtained from a plant according to claim 18.

25. Use of a greenhouse for growing plants comprising an atmosphere
wherein the content of a reactive oxygen species can be controlled to
prevent periodic oxidative stress during early ontogeny of said plants.

26. Use according to claim 25, wherein said reactive oxygen species is
O.sub.3.

27. Use according to claim 26, wherein said greenhouse is provided with
O3 scrubbers.

28. A method for optimizing the production of a specific type of plant on
a selected cultivated area, comprising the steps of: a) determining for
said specific type of plant the early ontogenic phase wherein the
baseline respiration rate or the rate of respiration via the alternative
oxidase (AOX) pathway is fixed to a permanent minimum level; b)
monitoring in the air over said cultivated area the concentration of
reactive oxygen species; and c) sewing a seed or planting a seedling or
plantlet of said specific type of plant and allowing the seedling or
plantlet to develop into a plant, wherein during said early ontogenic
phase of said plant an exposure to undesirable concentrations of reactive
oxygen species from the air over said cultivated area is essentially
prevented by selecting the moment of sewing or planting using the data
obtained in step b).

29. An agrobiological composition comprising isoprene emitting
microorganisms, said composition further comprising growth nutrients for
said microorganism and substances that induce isoprene production in said
microorganism, wherein said microorganism is preferably a transgenic
microorganism comprising a vector for the expression of isoprene
synthase, operably linked to a promoter functional in said microorganism.

30. An agrobiological composition comprising catalase-producing
microorganisms, said composition further comprising growth nutrients for
said microorganism and substances that induce catalase production in said
microorganism, wherein said microorganism is preferably a transgenic
microorganism comprising a vector for the expression of catalase,
operably linked to a promoter functional in said microorganism, and
wherein said microorganism is symbiotic to said plant.

31. Method for increasing the yield of a plant grown under conditions
that cause periodic oxidative stress in said plant, comprising
administering during the early ontogeny of said plant an agrobiological
composition comprising isoprene emitting microorganisms, said composition
optionally further comprising growth nutrients for said microorganism and
substances that induce isoprene production in said microorganism, wherein
said microorganism is preferably a transgenic microorganism comprising a
vector for the expression of isoprene synthase, operably linked to a
promoter functional in said microorganism.

32. Method for increasing the yield of a plant grown under conditions
that cause periodic oxidative stress in said plant, comprising
administering during the early ontogeny of said plant an agrobiological
composition comprising catalase-producing microorganisms, said
composition optionally further comprising growth nutrients for said
microorganism and substances that induce catalase production in said
microorganism, wherein said microorganism is preferably a transgenic
microorganism comprising a vector for the expression of catalase,
operably linked to a promoter functional in said microorganism, and
wherein said microorganism is symbiotic to said plant.

Description:

RELATED APPLICATIONS

[0001] This application is a continuation of PCT application number
PCT/NL2009/050348 designating the United States and filed Jun. 16, 2009;
which claims the benefit of U.S. patent application Ser. No. 12/139,753
and filed Jun. 16, 2008 both of which are hereby incorporated by
reference in their entireties.

FIELD OF THE INVENTION

[0002] The present invention is in the field of crop production. More in
particular, the present invention relates to methods for growing plants
and methods for increasing the average yearly crop production of a crop
production area. The invention further relates to plants produced by the
method of the invention exhibiting increased yield, and to greenhouses
comprising means for controlling the atmospheric content of a reactive
oxygen species.

BACKGROUND OF THE INVENTION

[0003] Global demand for wheat, rice, corn, and other essential grains is
expected to steadily rise over the next twenty years. Apart from serving
as a food source, the demands will rise as plant-based fuel and chemicals
production by biological processes is growing. Meeting this demand by
increasing production through increased land use is not very likely; and
while better crop management may make a marginal difference, most
agriculture experts agree that this anticipated deficit must be made up
through increased crop yields.

[0004] Modern crop production management systems are tailored to optimize
each and every parameter that influences crop yield. Management of
moisture and nutrient availability and control of pests is standard in
all agricultural production schemes for field crops. In greenhouse crops,
availability of CO2 and light and temperature may in addition be
optimized. Hence, most crops are currently produced under apparently
optimal conditions. Although some variation is perceived as "natural",
realistic production efficiencies are still a fraction of theoretical
maximum levels. In fact, year-average efficiency levels attained in the
field for most crops are often less than half of the maximum levels
observed in particular years. The reason for this difference is not
properly understood. Hence, there is a need for further understanding and
optimizing biological production efficiencies.

[0005] Plant biomass production is determined by the plants net carbon
fixation efficiency. This efficiency is governed by the rate of carbon
gain in terms of CO2 fixation by photosynthesis and the rate of
carbon loss in terms of CO2 emission by respiration. While gross
photosynthesis rises with temperature, so does respiration and whereas
the photosynthesis rate tends to flatten at the optimum of the
photosynthetic enzyme rubisco of about 25° C., respiration
continues to rise rapidly above this temperature and roughly doubles
every 10° C. (Q10 is usually 2). At temperatures above about
35° C. (at least for C-3 species) all the sugar produced is used
to support respiration. At temperatures higher than 35° C., plants
respire more sugar than they can produce which leads to deterioration and
ultimate death of the plant. Consequently the net photosynthesis (the
production of energy compounds minus their use by respiration including
photorespiration) must be considered when attempting to optimize
production. As noted above, traditional methods for improving carbon
balance are aimed at improving the gross photosynthesis rate. Although a
relationship between respiration rate and temperature is well known, and
respiration is known to vary depending on protein turnover and
maintenance requirements, baseline respiration rates are generally
believed to be relatively fixed.

[0006] During their life cycle, plants undergo a large number of
physiological, biochemical and morphological changes that are controlled
by alterations in gene expression. Yet, the morphogenetic capacity of
plants is a function of both genetic and environmental parameters, of
which thermal and light conditions appear to have the greatest influence.
Plant ontogeny, the sum of morphogenic processes that describe the
development of a plant from seed germination through to maturity, is
known to influence plant production parameters, such as the degree to
which plants compensate after defoliation or herbivore damage. Also, it
is known that factors that affect crop growth early in ontogeny often
produce modifications that extend through the season and may be manifest
in altered economic yield. It is believed that this is the result of
epigenetic factors, among which DNA-methylation is one of the best known.
Plant ontogeny includes developmental changes in plant architecture,
storage capacity, and resource allocation to different functions (e.g.,
storage, defence, reproduction). In the case of woody species, an
increase in the plant age is associated with changes in resource
allocation patterns, as the carbon/nutrient balance, storage capacity,
and access to water and nutrients usually increase, while root to shoot
ratio, growth rate, photosynthesis, stomatal conductance, hormone
production, and metabolic activity typically decrease. In addition,
morphological differences between juvenile and adult trees include
variation in leaf morphology, phyllotaxy, shoot orientation, seasonal
leaf retention, presence of adventitious roots, and leaf-specific mass.

[0007] It is however not known whether crop production in general is
influenced by ontogenic processes, or whether factors that affect crops
early in ontogeny can produce modifications that extend throughout the
life of the plant.

[0008] Isoprene (2-methyl-1,3-butadiene) is emitted by a large number of
plant species. Isoprene emission generally consumes a significant
percentage of the carbon fixed during photosynthesis. The role of
isoprene emission in plants is not fully understood. It was suggested to
provide protection against heat stress. Indeed, isoprene emissions in
some plants exhibit temperature response patterns that are dependent on
the plant's growth temperature. Other alkenes are also found to increase
thermotolerance in leaves. Isoprene is also suggested to provide a more
general protection against stress conditions and in particular against
(photo)-oxidative stress and it was recently shown to protect leaves
against ozone. The ability of alkenes, such as isoprene, to react with
singlet oxygen, ozone and OH radicals is well known, and isoprene
emission has been suggested as a quencher of ozone. Thus, although the
correlation between leaf temperature and isoprene emission in plants is
well known, the physiological role of isoprene emission, quite costly to
the plant, is still under debate.

[0009] Also vitamin C, ascorbate, is recognized as a factor that protects
plants against oxidatvive stress, such as ozone stress. Further, tartaric
acid and lycopene (a tetraterpene consisting of 8 isoprene units) have
been proposed as a plant-derived anti-oxidants.

[0010] It is however not understood how processes for the protection
against stress conditions can be made beneficiary so as to increase the
yield of said plant. Hence there exists a need for processes and plants
exhibiting increased yield despite the presence of yield lowering
processes that are essential to plant survival.

SUMMARY OF THE INVENTION

[0011] The present inventor has now discovered that baseline respiration
rate, in particular the AOX component of the total dark respiration, is
fixed early in the ontogeny of plants. In fact, the inventor has
discovered that oxidative stress early in the ontogeny of plants results
in plants having a high baseline respiration rate for the rest of their
productive life span. Although in mature plants, the respiration rate may
temporarily increase in response to altered environmental conditions, it
will return to a rate the level of which is ultimately fixed at an early
stage of development.

[0012] This discovery was done in Brazil, where annual sugar cane burnings
result in high temporary atmospheric concentrations of ozone. It was
found that plants that were planted in a period wherein ozone
concentrations were lowest consistently exhibited high production,
whereas plants that were planted in a period such that the early
ontogenic phase coincided with these adverse conditions consistently
exhibited low production rates.

[0013] Hence, based on these findings the inventor found that plants that
are capable of counteracting the adverse effects of atmospheric ozone or
that are prevented in any way from encountering these adverse effects
during early ontogeny exhibit high production, in terms of dry matter
content.

[0014] Early in ontogeny plants determine a default respiration rate based
on the conditions that prevail at that time in their life. An increase in
protein turnover, for instance due to increased tissue damage, will
result in a higher respiration rate. It was discovered that in early
ontogeny this is essentially a one-way street: the baseline respiration
rate can go up, but it cannot come down. As a result, a plant that is
exposed to unfavourable conditions such as high ozone concentrations that
result in leaf damage and increased respiration rates early in ontogeny,
will keep high baseline respiration rates essentially throughout its
life, or at least for prolonged periods of time. Hence, such a plant will
exhibit a low net production, as much of the assimilated carbon is
respired.

[0015] The present inventor has now discovered that plants that are grown
in the presence of ozone-protecting compounds, and hence that are
protected from the effects of ozone (i.e. oxidative stress), in
particular at a certain, early, stage in their life, show an enormous
increase in biomass production rates and on top of that a higher level of
disease-resistance. The presence of ozone-protecting compounds can be
achieved by providing specific climatological conditions in a greenhouse,
or by producing these compounds in the vicinity of the plant e.g. by
micro-organisms or by the plants themselves. The present inventors have
discovered that levels or emission rates of ozone-protecting compounds in
the vicinity of plants or in transgenic plants must be such that baseline
respiration rates, in particular the AOX component thereof, are not
elevated by ozone exposure. For isoprene, this is achieved when at least
during early ontogeny of the plants, the isoprene emission by the plant
at 30° C. is at least 20 nmolm-2s-1. Other suitable
minimal emission rates to avoid increase in the baseline respiration
rates, in particular the AOX component thereof, are for instance 5
μg/g dry weight/h, and 0.03 μg/cm2/h or 1.25
nmolm-2s-1.

[0016] The mitochondrial electron transport chain involves two pathways:
the cytochrome pathway and the alternative oxidase (AOX) pathway. In
plants, heat stress, and as contemplated herein also drought stress,
temperature stress, radiation stress, and salt stress induces the release
of cytochrome c (cyt-c) from the respiratory chain, which in turn leads
to elevated levels of Reactive Oxygen Species (ROS) and induction of AOX.
AOX is a cyanide-resistant, hydroxamic-acid-sensitive terminal oxidase
found in the inner mitochondrial membranes of plants, some fungi, and
trypanosomes. The activity mediates the non-proton-translocating transfer
of electrons from the UQ pool to molecular O2 to form water.
Electrons flowing through AOX bypass proton-translocating complexes III
and IV of the Cyt-mediated electron transport chain, causing the
oxidative potential energy to be lost as heat. The function of AOX in
plants during normal vegetative growth and development remains unclear.
The present inventor has now found that the minimum level of the OAX
pathway as component of the total dark respiration is determined early in
the life of the plant, that is: in the ontogenic phase, and that the
respiration efficiency in terms of ATP generated, and therefore the yield
of the plants, is less when the plants are exposed early in there life to
conditions that elevate the AOX pathway. It has now also been found that
this rise in the AOX pathway can be prevented intentionally, by
manipulating the plant or its environment during early ontogeny. Such a
manipulation is only possible on an effective level when appreciating the
present invention.

[0017] In a first aspect, the present invention provides a plant,
preferably grown under conditions that cause periodic oxidative stress,
wherein said plant or the environment of said plant was manipulated such
that an increase in the rate of respiration and/or protein turnover in
said plant due to said periodic oxidative stress was intentionally
prevented during the early ontogeny of said plant. For instance, the
plant may have been grown during its early ontogeny in the presence of a
sufficient amount of ozone-protecting compound in case the periodic
oxidative stress was caused by ozone. Preferably said ozone-protecting
compound is produced in the vicinity of the plant, by micro-organisms or
by the plant itself.

[0018] A plant of the present invention is characterized by the fact that,
although it was exposed to oxidative stress-inducing conditions during
its early ontogeny, exhibits an AOX as component of total dark
respiration that is essentially equal to a plant of the same variety that
has never been exposed to oxidative stress, and exhibits an AOX as
component of total dark respiration that is significantly lower (e.g. at
least 5-25% lower) than that of a plant of the same variety that was
exposed to said same oxidative stress-inducing conditions during its
early ontogeny but which plant or its environment were not manipulated to
prevent an increase in the AOX. Moreover, the plants have increased dry
matter content relative to an exposed plant which, or the environment of
which, was not manipulated as indicated herein.

[0019] In another preferred embodiment of a transgenic plant of the
present invention, said plant is from a species that does not naturally
emit isoprene. Such a plant is herein referred to as a plant from a
non-isoprene emitting plant species. Also contemplated are low-isoprene
emitters, which are referred to herein as plants of which the isoprene
emission rates are to be increased if an increase in the AOX during early
ontogeny as a result of periodic oxidative stress is to be prevented by
conversion of ozone.

[0021] In another preferred embodiment of the plant of the invention, said
early ontogeny is the period between germination and the generative
phase, thus preferably the vegetative phase, that is before the plants
induce and develop flowers. This stage can be anywhere between a few days
to 0.5-6 months post germination and is generally longer for trees.

[0022] In an alternatively preferred embodiment of a plant of the
invention said plant exhibits a baseline respiration rate (including and
preferably with reference to the alternative oxidase pathway) as the rate
of that is at most 80%, preferably at most 70, 60, 50, 40, 30, or even
20% of that of a plant which has not been subjected to ozone-protective
measures as described herein, at least during early ontogeny of said
plant. In a highly preferred embodiment this early ontogeny is the period
15-60 days after emergence (DAE) of the first leaf of said plant.
Alternatively, in another highly preferred embodiment this early ontogeny
is the period coinciding with the pre-floral stage of said plant.

[0023] In still another preferred embodiment of the plant of the
invention, said plant is from a species that does not naturally produce
an anti-oxidant compound mentioned above.

[0024] In still another preferred embodiment of the plant of the
invention, said plant is an adult plant, and said adult plant exhibits a
first rate of respiration under ambient ozone that is essentially equal
to the rate of maintenance respiration exhibited by said plant during
early ontogeny, and wherein said plant upon temporary exposure to ambient
plus 50 or 100 ppbv of ozone exhibits a second rate of respiration, which
second rate is a significant increase relative to said first rate, and
wherein following said temporary exposure said second rate of respiration
returns to pre-exposure levels.

[0025] In an even more preferred embodiment of this plant said rate of
maintenance respiration is the rate of respiration of a young full grown
leaf of a plant measured when said plant has not been exposed to ozone,
preferably said rate of respiration refers to the AOX component in de
total (dark) respiration due to oxidative stress as a result of which the
respiration loses efficiency in terms of generating ATP. Thus, preferably
the AOX in a plant of the invention that has passed the ontogenic phase
and that has been exposed to AOX-enhancing levels of ozone, is close to
zero, or at least close to that of a plant never exposed to ozone or
other oxidative stress. Thus, plants of the invention when placed in an
oxidative-stress-free environment have the ability of having their AOX
essentially return to 0% (or pre-exposure levels). Whereas plants that
have not been grown by a method of the invention and that have been
exposed to oxidative stress during the ontogenic phase have relatively
high AOX components even when thereafter placed in an
oxidative-stress-free environment.

[0026] In another aspect, the present invention provides a method for
increasing the yield of a plant grown under conditions that cause
periodic oxidative stress in said plant, comprising the step of growing a
seedling, tissue culture or plantlet into a plant, and further comprising
the step of manipulating said plant or the environment of said plant such
that an increase in the rate of respiration and/or protein turnover in
said plant due to said periodic oxidative stress is intentionally
prevented during the early ontogeny of said plant.

[0027] A method for increasing the yield of a plant includes reference to
methods for increasing stress tolerance as indicated herein and to
methods for increasing disease resistance in said plant.

[0028] In embodiments of this method, the oxidative stress is the stress
in a plant that may be brought about by radiation, heat, salt, drought,
SO2, low CO2, or reactive oxygen species, including
H2O2, or O3, preferably H2O2 or O3, most
preferably O3 or precursors of O3, such as NOx.

[0029] In another preferred embodiment of the method of the invention, the
oxidative stress is prevented by controlling the content of a reactive
oxygen species (ROS) in the growth environment. Said reactive oxygen
species in the growth environment may be H2O2, or O3,
preferably H2O2 or O3, and is most preferably O3 in
the growth atmosphere. The ROS content may be controlled in one of many
ways. For instance in a closed system, it may be controlled by using ROS
scrubbers. A suitable ROS scrubber is for instance an activated carbon
filter. Alternatively, the ROS and particularly the O3 content in
the growth atmosphere may be controlled by exposing the plant or its
growth atmosphere to isoprene. Isoprene is known to neutralize ozone. In
a further preferred embodiment, the isoprene may be produced by
micro-organisms (including transgenic or recombinant micro-organisms
transformed with a gene encoding the enzyme isoprene synthase) in the
vicinity of the plant. Alternatively, increased levels of CO2 may be
used to counteract the effects of oxidative stress during the ontogenic
phase.

[0030] In yet another preferred embodiment of the method of the invention,
the plant is a perennial plant and/or a crop plant, preferably a
protected crop plant.

[0031] In another aspect, the present invention provides a method for
increasing the average yearly crop production of a crop production area,
comprising growing a plant according to a method of the invention as
described above and using said plant as a crop plant in said production
area.

[0032] In a preferred embodiment of this method, the increase in the
average yearly crop production is brought about by an improved net carbon
fixation efficiency, biomass production, dry matter content and/or pest
resistance of said crop plant.

[0033] In yet another aspect, the present invention provides a plant part
obtained from a plant of the present invention as described above.

[0034] In still a further aspect, the present invention provides a
greenhouse for growing plants comprising an atmosphere wherein the
content of a reactive oxygen species is controlled. Preferably, said
reactive oxygen species is O3 and the greenhouse of the present
invention is therefore preferably provided with O3 scrubbers optionally
in combination with O3 precursor transformers (e.g. a light source
transforming NO2 into NO+O3).

[0035] In yet another aspect, the present invention provides a method for
optimizing the production of a specific type of plant on a selected
cultivated area, comprising the steps of: [0036] a) determining for
said specific type of plant the early ontogenic phase wherein the
baseline respiration rate is fixed to a permanent minimum level; [0037]
b) monitoring in the air over said cultivated area the concentration of
reactive oxygen species, for instance by using an atmospheric sensor
capable of determining the concentration of reactive oxygen species in
air; and [0038] c) sewing a seed or planting a seedling or plantlet of
said specific type of plant and allowing the seedling or plantlet to
develop into a plant, wherein during said early ontogenic phase of said
plant an exposure to undesirable concentrations of reactive oxygen
species from the air over said cultivated area is essentially prevented
by selecting the moment of sewing or planting using the data obtained in
step b).

[0039] In another aspect, the present invention provides an agrobiological
composition comprising isoprene emitting microorganisms and an
agronomically acceptable carrier, said composition optionally further
comprising growth nutrients for said microorganism and substances that
induce isoprene production in said microorganism, wherein said
microorganism is preferably a transgenic microorganism comprising a
vector for the expression of isoprene synthase, operably linked to a
promoter functional in said microorganism.

[0040] In another aspect, the present invention provides an agrobiological
composition comprising catalase-producing microorganisms and an
agronomically acceptable carrier, said composition optionally further
comprising growth nutrients for said microorganism and substances that
induce catalase production in said microorganism, wherein said
microorganism is preferably a transgenic microorganism comprising a
vector for the expression of catalase, operably linked to a promoter
functional in said microorganism, and wherein said microorganism is
symbiotic to said plant.

[0041] In another aspect, the present invention provides a method for
increasing the yield of a plant grown under conditions that cause
periodic oxidative stress in said plant, comprising administering during
the early ontogeny of said plant the agrobiological composition of the
present invention.

[0046] The term "plant," as used herein, refers to any type of plant. The
inventors have provided below an exemplary description of some plants
that may be used with the invention. However, the list is provided for
illustrative purposes only and is not limiting, as other types of plants
will be known to those of skill in the art and could be used with the
invention.

[0052] "Plant tissue" includes differentiated and undifferentiated tissues
or plants, including but not limited to roots, stems, shoots, leaves,
pollen, seeds, tumor tissue and various forms of cells and culture such
as single cells, protoplast, embryos, and callus tissue. The plant tissue
may be in plants or in organ, tissue or cell culture.

[0053] The term "perennial", as used herein, refers to a plant that lives
for more than two years. Perennial plants can be short-lived (only a few
years) or they can be long-lived, as some woody plants, such as trees.

[0054] The term "crop plant", as used herein, refers to a plant which is
harvested or provides a harvestable product. Particularly preferred
plants for use in aspects of the invention are protected (greenhouse)
crop plants.

[0055] The terms "seedling" and "plantlet", as used herein, are
interchangeable and refer to the juvenile plant grown from a sprout,
embryo or a germinating seed and generally include any small plants
showing well developed green cotyledons and root elongation and which are
propagated prior to transplanting in the ultimate location wherein they
are to mature.

[0056] The term "tissue culture", as used herein, refers to a culture of
plant cells wherein the cells are propagated in a nutrient medium under
controlled conditions.

[0057] "Significant increase" is an increase that is larger than the
margin of error inherent in the measurement technique, preferably an
increase by about 10%-50%, or even 2-fold or greater.

[0058] "Significantly less" means that the decrease is larger than the
margin of error inherent in the measurement technique, preferably a
decrease by about 2-fold or greater.

[0059] The term "early ontogeny", as used herein, refers to the early
phase in the course of growth and development of an individual to
maturity (the ontogenic phase). This phase will differ in length between
plant species. For roses, this period is estimated to amount to about 6
to 8 months. In essence, the early ontogenic phase terminates when the
plant characteristics in response to environmental stimuli, such as
respiration rate, become established and fixed. The skilled person can
determine the early ontogenic phase for any plant species by measuring
the ozone-induced respiration rate in plants at periodic intervals and
determining at what age of the plant the increase in respiration rate due
to ozone exposure does not return to pre-exposure values, but is fixed.
The ontogenic phase for many plants will be the development of the
seedling trough the vegetative stage and will generally end upon entry of
the generative stage (floral stage).

[0060] The term "manipulating said plant" refers to a technical
intervention in said plant, inter alia by genetic manipulation, for
instance by providing a transgenic plant as described herein, such that
said plant does not increase its baseline respiration rate despite the
presence of oxidative stress-inducing factors such as stress-causing
ozone levels.

[0061] The term "manipulating the environment of said plant" refers to a
technical intervention in the surroundings or ambient of said plant,
inter alia by lowering the stress to said plant, for instance by
providing an environment wherein the oxidative stress-inducing factors
such as stress-causing ozone is reduced by technical intervention to
sub-stress levels, or at least to such levels that said plant does not
increase its baseline respiration rate relative to the situation without
said intervention where, in the presence of oxidative stress-inducing
factors such as stress-causing ozone levels, said plant would have
increased its baseline respiration rate.

[0062] The term "intentionally" in the context of intentionally prevented
an increase in the rate of respiration and/or protein turnover in said
plant refers to the performance of an activity such as a technical
intervention or a lack thereof as described above, with the purpose of
maintaining the AOX component of the total dark respiration in said plant
as defined herein at levels essentially equal to that of a plant that has
never been exposed to oxidative stress, to thereby achieve an increase in
the yield of said plant.

[0063] The term "epigenetic", as used herein, refers to the state of the
DNA with respect to heritable changes in function without a change in the
nucleotide sequence. Epigenetic changes can be caused by modification of
the DNA, in particular chromatin remodeling caused by modifications of
the histone proteins and DNA methylation. These changes affect gene
transcription and ultimately affect phenotype. Epigenetic changes thus
involve factors that influence behavior of a cell without directly
affecting its DNA or other genetic components. The epigenetic view of
differentiation is that cells undergo differentiation events that depend
on correct temporal and spatial repression, derepression, or activation
of genes affecting the fate of cells, tissues, organs, and ultimately,
organisms. Thus epigenetic changes in an organism are normal and result
in alterations in gene expression. For example, epigenetic transformation
of a normal cell to a tumor cell can occur without mutation of any gene.

[0064] The term "respiration", as used herein, refers to the process by
which O2 is transported to and used by the cells and CO2 is
produced and eliminated from the cells during which process organic
matter is oxidized.

[0065] The term "protein turnover", as used herein, refers to the flow of
amino acids from existing protein into newly synthesized protein. Protein
turnover is generally regarded as one of the most important maintenance
processes in plants in terms of energy requirements. Both biosynthetic
and breakdown processes affect the rate of protein turnover. Both protein
synthesis and protein degradation require respiratory energy. Protein
turnover has several important functions in regulating the plant's
metabolism. Together with protein synthesis, degradation is essential to
maintain appropriate enzyme levels and to modulate these levels based on
internal and external signals. Furthermore, protein degradation is
important in allowing a plant to cope with changing environmental
conditions. When nutrients become limiting, the rate of protein turnover
is accelerated by increasing the rate of degradation relative to
synthesis, which generates a pool of free amino acids from less essential
proteins that can be used to assemble more essential ones.

[0066] The term "radiation", as used herein, includes both particle
radiation (ie electrons, protons), high-energy electromagnetic radiation
(i.e. x-rays, gamma rays) and other ionizing radiation in the
radiant-energy spectrum, as well as non-ionizing electromagnetic
radiation, and radiation in the ultraviolet, visible light, and infra-red
spectrum.

[0067] The term "periodic" in the context of "periodic stress" refers both
to a discontinuous form of stress that is experienced by said plant for
interrupted periods, as well as to a form of stress that is experienced
by said plant for uninterrupted periods (e.g. permanently) but at varying
level.

[0068] The term "oxidative stress", as used herein, refers to the state in
which cells are exposed to excessive levels of molecular oxygen or
reactive oxygen species (ROS) to the extent--ultimately--that damage is
incurred and cellular repairs systems are mobilized. Before damage is
done however, the plant cells anticipating risk to damage will upscale
certain processes (e.g. by protein turnover) and/or down scales other
processes (e.g. like cytoplasmic streaming) to prevent damage from
occurring. This also qualifies as oxidative stress Oxidative stress may
thus be measured by increased protein turnover rates. As used herein,
oxidative stress may inter alia be the result of drought stress,
temperature stress, radiation stress, heat stress, salt stress, or
reactive oxygen species. Oxidative stress as defined herein is therefore
the process that increases the AOX pathway. Preventing the plant to
increase its AOX pathway during the early ontogeny therefore renders such
a plant less sensitive to drought stress, temperature stress, radiation
stress, heat stress, salt stress. Hence methods of the present invention
inherently have the result of rendering plants more resistant to other
stresses than oxidative stress, including resistance to pests and
disease.

[0069] The term "reactive oxygen species" (ROS), as used herein, refers to
oxygen ions, free radicals, and peroxides, both inorganic and organic.
They are generally very small molecules and are highly reactive due to
the presence of unpaired valence shell electrons. ROSs form as a natural
by-product of the normal metabolism of oxygen and have important roles in
cell signalling. However, during times of environmental stress ROS levels
can increase dramatically, which can result in significant damage to cell
structures. This cumulates into a situation known as oxidative stress.

[0070] The term "ambient ozone" is used herein to indicate an ozone level
of about 10 to 15 ppbv to about 35 ppbv. The level may vary between night
and day and may be about 0 ppbv at night and about 45 to 120 ppbv during
the day.

[0071] The term "growth environment", as used herein, refers to the soil,
substrate, solution or air in which the plant is growing.

[0072] The term "scrubber", as used herein with reference to reactive
oxygen species or O3 (ozone) scrubber, refers to a system, device,
or chemical capable of binding or chemically converting reactive oxygen
species, such as O3 to the effect that said reactive oxygen species
is eliminated from the environment which is placed in contact with the
scrubber.

[0073] The term "isoprene", as used herein, refers to the chemical
compound 2-methylbuta-1,3-diene.

[0074] The term "lycopene", as used herein, refers to the chemical
compound (6E,8E,10E,12E,
14E,16E,18E,20E,22E,24E,26E)-2,6,10,14,19,23,27,31-octamethyldotriaconta--
2,6,8,10, 12,14,16,18,20,22,24,26,30-tridecaene, and is a tetraterpenoid
compound, assembled from 8 isoprene units.

[0075] The term "endogenous" as in "endogenously produced" refers to
produced within the plant (cell).

[0076] The term "isoprene synthase", as used herein, refers to the enzyme
with registry number EC 2.5.1.-, that catalyses the elimination of
pyrophosphate from dimethylallyl diphosphate to form isoprene. The amino
acid sequence of the enzyme in Populus alba is provided in SEQ ID NO:1.
The term is considered to cover homologues having >80%, preferably
>90% amino acid identity with SEQ ID NO:1, and in particular refers to
homologues that produce isoprene from dimethylallyl diphosphate.

[0077] The term "prefloral stage", as used herein, refers to the ontogenic
stage preceding the emergence of reproductive structures in a plant.

[0078] The term "post-germination", as used herein, refers to the period
in the development of the plant following the emergence of the radicle
from the seed.

[0079] The term "average yearly crop production", as used herein, refers
to the amount of crop produced per annum or season (average seasonal crop
production) in the form of weights or number of plants or plant parts
harvested or weight gain in crop biomass (fresh and or dry weight) are
expressed per unit of production area, and wherein the individual amounts
per annum for multiple years are summated and divided by the number of
years.

[0080] The term "production area", as used herein, refers to a location
where plants are grown and where products in the form of plants or plant
parts are produced for harvest. The size of the production area is
generally expressed in square meters or acres of land. A production area
can be an open field or a greenhouse.

[0081] The term "net carbon fixation efficiency", as used herein, is used
interchangeable with the term "net photosynthetic efficiency" and refers
to the net efficiency with which carbon dioxide is converted into organic
compounds, taking into account the losses due to respiration.

[0082] The term "biomass production", as used herein, refers to the
production of plant derived organic material.

[0083] The term "dry matter content", as used herein, refers to the mass
fraction (%) that remains after the water fraction (%) has been removed
by drying.

[0084] The term "pest resistance", as used herein, refers to resistance
against viral, bacterial, fungal, and insect pests, as well as pests by
molluscs and nematodes.

[0085] The term "greenhouse", as used herein, refers to any structure
comprising walls, a roof, and a floor designed and used principally for
growing plants in a controlled and protected environment. The walls and
roof are usually constructed of transparent or translucent material to
allow passage of sunlight for plant growth.

Methods of Growing Plants

[0086] The methods of growing plants according to the present invention
are based on three essential realizations:

i) respiration rates reached in a plant during early ontogeny culminate
in the development of a baseline respiration rate, which is the minimum
rate at which said plant will respire when mature; ii) this baseline
respiration rate is attained by one or more step-up increments and is
ultimately fixed at baseline level by epigenetic changes; iii) the
step-up increments in the respiration rate are the plant's response to
increased protein turnover rates which in turn are the result of
oxidative stress experienced by said plant, and iv) the step-up
increments in the AOX component of the total dark respiration are the
result of oxidative stress experienced by said plant.

[0087] Therefore, when attempting to keep the respiration rates in mature
plants as low as possible, it is important to minimize the oxidative
stress to said plant during the phase wherein the baseline respiration
rate is fixed by epigenetic factors, that is, early in ontogeny.

[0088] Hence, a method of growing plants according to the present
invention comprises the important step of allowing a seedling, tissue
culture or plantlet to develop into a plant and to pass through its early
ontogenic phase wherein epigenetic factors fix the baseline respiration
rate to a permanent minimum level. The early ontogenic phase wherein
epigenetic factors fix the baseline respiration rate to a permanent level
can differ for different plants, and can therefore not be defined for
each and every plant species in advance. Also, the phase at which
epigenetic factors determine the permanent minimum rate of respiration
may vary between plant varieties within a species. This phase may however
be experimentally determined for each and every plant species or plant
variety as follows:

[0089] Seedlings, tissue cultures or plantlets are allowed to develop into
mature plants. The total development time is recorded and divided into a
large but practical number of regular intervals (between 2 and for
instance 10, 20, 50, 100, or 1000 intervals). During each interval, the
respiration rate of the developing plant is measured using standard
techniques. After each measurement, the plant is temporarily subjected to
oxidative stress, for instance by exposing it to ozone. The oxidative
stress can optionally be applied at incrementally increasing levels
during the different intervals. The early ontogenic phase wherein
epigenetic factors fix the baseline respiration rate to a permanent level
is now determined from the data obtained as:

a) the total number of intervals preceding the interval in which the
respiration rate returns to a baseline level after said oxidative stress
period, or b) the total number of intervals during which the respiration
rate shows a step-wise increase relative to the preceding interval and no
decrease in the subsequent or following interval.

[0090] Roughly, in a method of the present invention the early ontogenic
phase wherein epigenetic factors fix the baseline respiration rate to a
permanent level is the prefloral stage, preferably a period from 0.1-6
months, preferably 0.5-6 months following the germination.

[0091] A method of growing plants according to the present invention
further comprises the important step of preventing during early ontogeny
of said plant the occurrence of an undesirable increase in the rate of
respiration and/or protein turnover due to oxidative stress. It is to be
understood that in accordance with the above-described model for
epigenetic fixation of baseline respiration rates, an undesirable
increase in the rate of respiration is equivalent to an undesirable
increase in the rate of protein turnover.

[0092] Oxidative stress is often, but not exclusively, the result of
damage to the plant brought about by radiation or reactive oxygen
species. Hence, the prevention of an undesirable increase in the rate of
respiration and/or protein turnover due to oxidative stress can be
attained by preventing exposure to radiation or reactive oxygen species
that result in such an increase. Most notably, intracellular
H2O2 levels and atmospheric O3 concentrations are
maintained at sub-stress levels. Such levels may differ between plant
species and between plant varieties, but can be easily determined
experimentally. For instance, a plant can be exposed to a certain test
level of radiation or reactive oxygen species for a predetermined period
of time (i.e. varying between several minutes to several weeks) and the
rate of respiration and/or protein turnover is determine before and after
said exposure. A sub-stress level of radiation or reactive oxygen species
is the level at which the exposure does not result in an increase of the
post-exposure rate relative to the rate before.

[0093] Thus, in preferred embodiments of methods of the present invention,
the oxidative stress is prevented by controlling the content of a
reactive oxygen species in the growth environment such that they remain
at sub-stress levels.

[0094] The growth environment may be the soil, substrate, medium or
atmosphere in which the plant is grown. Preferably the content of the
reactive oxygen species, most preferably O3, is controlled in the
growth atmosphere.

[0095] In order to prevent oxidative stress, the O3-content of the
growth atmosphere of the plant during the early ontogenic phase is
preferably kept below 100 ppbv, most preferably below 75 ppbv, even more
preferably below 60 ppbv, still more preferably below 50 ppbv, even more
preferably below 40 ppbv, yet even more preferably below 35, 30, 25, 20,
15, 10, 5 or 1 ppbv, wherein ppbv refers to parts per billion by volume.
Sporadic increases are likely not to have a major effect on the plants,
yet values above 100 ppbv should essentially be avoided at all times
during the early ontogenic phase.

[0096] Essentially, this may be attained in different ways. For instance,
the content in the total airspace (atmosphere) in which the plant is
grown may be controlled. Control in the total airspace will generally
involve the use of closed cultivation systems, such as greenhouses.
Effective control of the level of reactive oxygen species can be obtained
by the use of scrubbers or air filtering systems. Such filtering systems
are well known in the field of conditioning of air. Suitable scrubbers or
filter materials include those materials that absorb or adsorb reactive
oxygen species. An example of a suitable material is activated carbon,
but other materials capable of filtering reactive oxygen species from air
that is passed through the filter of over the filter material are also
suitable. It is preferred that an air circulation system is used wherein
the air is moved over or through the scrubber. Also suitable is the use
of a material that reacts with the reactive oxygen species in the
atmosphere, thereby rendering the reactive oxygen species unreactive. A
suitable example of such a material is isoprene. Hence, a greenhouse may
periodically be loaded with isoprene gas in order to remove reactive
oxygen species from the air. Generally, an amount of isoprene equivalent
to 1-1000, more preferably 50-150 parts per billion by volume (ppbv) of
air is sufficient.

[0097] Alternatively, only the content in the air that is in direct
contact with the plant surface may be controlled. This is most
effectively attained by providing the plant with an exogenous source of
isoprene. To this end, isoprene may be provided in the form of a vapour
in the growth atmosphere of the plant, or at least at the leaves of a
plant. Alternatively, isoprene may be produced by epiphytes such as
microorganisms including bacteria, fungi and yeasts living in symbioses
with the plant at its surface (leaf, stem or root).

[0098] Alternatively, only the content in the air in the endogenous free
space (that also difuses out into to the atmosphere) may be controlled,
e.g. by endophytes such as microorganisms including bacteria, fungi and
yeasts living in symbioses within the plant.

[0099] Alternatively, only the ROS content, specifically H2O2,
in the endogenous liquid free layer may be controlled, e.g. by
catalase-positive endophytes, such as microorganisms including bacteria,
fungi and yeasts living in symbioses within the plant and being capable
of liberating the H2O2 converting enzyme catalase.

[0100] Methods of growing plants according to the present invention have
as an advantage that the net biosynthetic efficiency of said plant is
increased during its entire lifetime. The invention is therefore
especially important for plants that have a life cycle of multiple years,
because it is in these plants that a high baseline respiration rate
attained during early ontogeny has the most impact on the total
production of the plant over its entire lifetime. Hence a plant in
aspects of the present invention is preferably a perennial plant.

[0101] It will also be appreciated that the methods of the present
invention are particularly suitable for plants in which production
parameters are very important, such as for instance in crop plants and
plants projected to help counteracting atmospheric CO2 accumulation
for climate stabilizing purposes (carbon credits).

[0102] A method of growing a plant according to the present invention is
preferably practiced on a large number of plants simultaneously as a part
of crop production. Hence, in another aspect, the present invention
provides a method for increasing the average yearly crop production of a
crop production area, comprising growing a plant of the invention or
according to a method of the invention as described above and using said
plant as a crop plant in said production area.

[0103] It is an advantage of the plants and methods of the present
invention that the average yearly crop production is no longer adversely
affected by seasonal fluctuations in the atmospheric load of reactive
oxygen species.

[0104] A crop production area can be a small field of a few square metres,
but most preferably is a large production area covering several acres or
even a large number of square miles. The advantages of the present
invention are best appreciated when a large number of plants are
involved. The methods of the present invention can be performed on large
cultivated areas, and can for instance comprise the monitoring of
environmental factors that influence oxidative stress, and selecting the
planting or sewing season such that in its early ontogenic phase as
defined herein the plant is exposed to a minimum of oxidative stress.
Thus, the method of the invention can be made part of a crop cultivation
system comprising the monitoring of for instance atmospheric reactive
oxygen species, and using this information to select the optimal planting
or sewing season for the crop such that the crops are not exposed to
undesirable or stress-level concentrations of atmospheric reactive oxygen
species such as O3.

[0105] Based on the teachings of the present invention, the skilled person
will understand that certain planting seasons in Brazil and other parts
of the world that suffer from high ozone levels during certain parts of
the year, are best avoided due to excessive atmospheric concentrations of
reactive oxygen species, such as during sugar cane burning. It is an
aspect of the present invention that such avoidance can be managed
effectively by monitoring the concentration of reactive oxygen species in
the air over a cultivated area using atmospheric pollution sensors or
"sniffers" for determining the concentration of reactive oxygen species
in air.

[0106] Therefore in another aspect, the present invention provides a
method for optimizing the production of a specific type of plant on a
selected cultivated area, comprising the steps of:

a) determining for said specific type of plant the early ontogenic phase
wherein epigenetic factors fix the baseline respiration rate to a
permanent level, for instance by using methods as described above; b)
monitoring for said selected cultivated area the concentration of
reactive oxygen species in the air over said cultivated area, for
instance by using an atmospheric sensor capable of determining the
concentration of reactive oxygen species in air, or sampling air and
determining the concentration in said sample; and c) sewing a seed or
planting a seedling or plantlet of said specific type of plant and
allowing the seedling or plantlet to develop into a plant, wherein during
early ontogeny of said plant an increase in the rate of respiration
and/or protein turnover due to oxidative stress as a result of reactive
oxygen species in the growth atmosphere is essentially prevented, by
selecting the moment of sewing or planting using the data obtained in
step b).

[0107] It should be understood that the monitoring in step b) need not be
constant but must be frequent enough to allow the determination of the
optimal moment for sewing or planting. The optimal moment for sewing or
planting is the moment that assures that the subsequent early ontogenic
phase wherein the baseline respiration rate is fixed to its minimum level
coincides with the lowest oxidative stress induction, such induction
being the result of the minimum sum of all oxidative stress inducing
factors present including the concentrations of reactive oxygen species
in the growth atmosphere.

[0108] When reference is made herein to terms such as optimizing the
production or increase in the average yearly crop production, it is meant
that such attributes include or are brought about by an improved net
carbon fixation efficiency, biomass production, dry matter content and/or
pest resistance of said crop plant.

[0109] Another aspect of the present invention is a greenhouse for growing
plants, in particular by methods of the present invention. The greenhouse
of the present invention comprises means for controlling the content of a
reactive oxygen species in the greenhouse atmosphere. Preferably, said
greenhouse is equipped with a scrubber for removing reactive oxygen
species from the growth environment. In addition, the greenhouse may be
equipped with systems or devices for monitoring the concentration of
reactive oxygen species in the greenhouse atmosphere (i.a. the air inside
the greenhouse), for instance in the form atmospheric sensors capable of
determining the concentration of reactive oxygen species in air.

EXAMPLES

Example 1

[0110] As one example, the present invention may be performed as follows.
In a greenhouse roses are grown under normal conditions of temperature,
water, nutrients and light. In addition, the greenhouse atmosphere is
controlled for the amount of ozone, for instance by using an ozone
scrubber in order to reduce the amount of ozone in the greenhouse
atmosphere. Furthermore, in order to avoid entry of ozone from the
outside atmosphere, the greenhouse is well closed.

[0111] At least for the duration of the early ontogenic period as defined
herein above, the ozone levels in the greenhouse are maintained at the
lowest possible levels, preferably below 20-30 ppbv. Thereafter, that is
after the early ontogenic phase, the plants may be grown under less
stringent conditions or the plants may be brought outside the greenhouse
for further growth.

[0112] The exposure to the beneficial growth environment will result in
plants having a lower respiration rate when mature--compared to plants
being exposed to high ozone environments, and such plants will exhibit
higher production output as described herein above.

Example 2

[0113] The Examples as described below demonstrate that the ontogenic
phase of plants as described herein provides for a memory effect with
respect to respiration via the AOX pathway. In particular it will be
shown that the ontogenic phase is a learning period during which
oxidative stress, such as provoked by ozone, irreversibly upregulates the
non ATP rendering Alternative Oxidative Pathway or AOX component within
the total dark respiration in ambients with higher ozone levels.
Respiration via this AOX pathway is the "alternative oxidative pathway
respiration", which is also referred to as "alternative path respiration"
(APR), "cyanide insensitive respiration" or "SHAM sensitive respiration".

[0114] The tests, which were carried out in 3 ambients with different
ozone levels, show that cotton plants at the end of their vegetative
phase and upon entering the generative phase (i.e. in the ontogenic
phase) have a defined and fixed maximum respiration efficiency expressed
as [TDR-AOX]/[TDR], wherein TDR is the total dark respiration, and
wherein AOX is the alternative oxidative pathway (AOX) respiration.

[0115] This respiration efficiency is set by the amount or level of ozone
to which the plant is exposed in its ontogenic phase, herein referred to
as the ontogenic phase level of ozone (OPLO). After the ontogenic phase,
exposure of the plants to ozone levels higher than OPLO increases the AOX
(AOX follows and upregulates). When the ozone level is subsequently
returned to OPLO, the AOX also returns to the initial ontogenic level.
Exposure of the plants to ozone levels lower than OPLO does not result in
a lowering of the AOX (i.e. the AOX doesn't follow but maintains it's
original level established during the ontogenic phase).

[0116] It was found that plants with higher respiration efficiency also
have higher leaf weight (both when expressed as fresh and as dry weight)
per surface area.

[0118] The seeds were planted in standard PE plastic flower vases no. 15
(height 15 cm, upper internal diameter of 15.5 cm, bottom internal
diameter of 12 cm, with eight 10 mm drainage holes in the bottom). At the
bottom of each vase a round piece of aquarium filter cloth of 12 cm
diameter was placed in order to prevent gravel stones to wash out of the
pot. 480 ml of washed stone gravel has been placed on top of the filter
cloth. A second piece of filter cloth was placed on top of the gravel in
order to separate it from the next layer above, and prevent the mixing of
the two layers. The second layer consisted of 1.30 litres of a mixture of
perlite and vermiculite (50/50% v/v), pre-saturated with 570 ml of
nutrient solution (see below).

[0119] For all crops except sugar cane 8 selected seeds were planted per
pot at the following depths: Cotton: 2 cm; beans: 2 cm; corn: 4 cm; rice:
1.5 cm; soy: 3 cm depth; wheat 2.5 cm. All pots were covered with a dome
consisting of a transparent (PP) 1 litre vase turned upside down. The rim
of this cover vase was neatly in contact with the inner wall of the
planting pot. The seeds were allowed to germinate by placing the pots in
the dark at 30° C. Cotton, beans and corn germinated first and
were placed inside a phytotron three days after planting of the seeds and
domes were removed the next day. Rice, soy and wheat germinated later and
were placed inside a phytotron 5 days after planting with removal of the
domes the next day.

[0120] For sugarcane 4 plantlets per pot were provided and these plantlets
were transplanted from the agar following washing of their root system
with nutrient solution (see below) in order to remove residual agar.
Every sugar cane pot was provided with a dome consisting of a 5 litre
water flask (PP) of which the bottom was removed creating a 30 cm high
dome. Crepe-tape was used to provide an air-tight connection between the
bottom rim of the flask and the planting pot. The 40 mm screw cap on top
functioned as a ventilation valve, initially maintaining near 100% RV to
prevent the plantlets from drying out. Directly after transplanting, the
sugar cane plantlets were placed in a phytotron (4 pots per phytotron)
more in particular in acrylic cabinets created therein (see below). The
sugar cane domes were removed 10 days after transplanting.

Nutrient Solution.

[0121] A single formulated nutrient solution was given to all crops during
the full testing period containing 15 meq/l cations and 15 meq/l anions
giving it an EC of 1.7 mS/cm. pH=5.8. Macro nutrients: K=4.50 mmol/l,
Ca=3.75 mmol/l, Mg=1.50 mmol/l, N--NO3=9.75 mmol/l, N--NH4=0 mmol/l,
SO4=1.95 mmol/l, PO4=1.35 mmol/l. Micro nutrients: B=28 μmol/l,
Zn-EDTA=13 μmol/l, Cu-EDTA=1.6 μmol/l, Mo=0.1 μmol/l,
Fe-EDDHMA=52 μmol/l, Mn-EDTA=12 μmol/l. On all instances where
water was required only distilled water was used. The nutrient solution
was the only water source for the plants.

Growth Conditions

[0122] Phytotron. In order to create and maintain the desired climate in
an ambient that physically supports the plants to be exposed and tested,
three identical phytotrons were constructed. Each phytotron was designed
in order to internally control: light level, air temperature, air
humidity, air CO2 level, air O3 level and air circulation.
Steel plated, electrostatically painted white modules with 50 mm and 100
mm thick isopore insulation designed for cold store construction were
used to create three identical and hermetically sealed rooms of 4.0 m by
3.0 m by 2.5 m (l×w×h)=30 m3. Each room was equipped
with a single sealed door for access.

[0123] Acryl Growing Cabinets. Inside each phytotron two fully transparent
growing cabinets (90 cm (deep)×120 cm (wide)×140 cm (height))
were provided (5-6 mm transparent acrylic plates). Each growing cabinet
was further divided into four plant-compartments with acrylic plates,
each plant compartment housing four pots with test plants. Each
plant-compartment was vertically separated from a neighbouring
compartment by a lamp-compartment housing 4 tube lights (Philips TLD 30
W/75, 2000 lumen & 25 μmol/s) vertically positioned one above the
other. The outside of the outermost plant-compartments was also fitted
with a lamp-compartment for complete illumination of the compartment. A
total of 40 Philips TLD 30 W/75 lamps were installed in each phytotron.
Each plant-compartment was further fitted with four port holes
(22×45 cm) that could be closed. The cabinets were positioned on a
metal base giving them a ground clearance of 60 cm. The top of each
cabinet was provided with a mirror facing inward to reflect upgoing
light.

[0124] Light Control: A Programmable Lighting Control (PLC) (model Logo
12/24 RC from Siemens) was used to control the photoperiod and lamp
sequence. The acrylic cabinets were equipped with four levels of light
counting 10 lamps each (40 lamps in total), numbered 1-4 from top to
bottom. Level 1 was activated at 5:00 a.m. In sequence every next level
came in one hour later providing full light at 8:00 a.m. At 8:00 p.m. all
levels were deactivated at once providing full darkness.

[0125] Anti-chamber. A sealed central corridor gave access to the
phytotrons and had the function of anti-chamber. The central corridor was
kept at negative pressure in order to minimize air mixing between the
phytotrons in the event of opening their doors. A blower fan at the end
of the corridor was used to further minimize ozone translocation from
high ozone to low ozone regime climates by moving the air from the lower
ozone chambers to the higher ozone levels (e.g. from phytotron 1 (set
point=0 ppbv O3) towards phytotron 2 (set point=30 ppbv O3)
towards phytotron 3 (set point=60 ppbv O3)).

[0126] CO2 Control: One central CO2 interface & control system
for CO2 control was used (C-Control II, Conrad Electronic GmbH,
Germany).

[0127] At 10 minutes interval the CO2 concentration in each of the
three phytotrons as well as in the outside ambient air was measured using
a CO2 analyzer (Priva 250E, (NDIR) gas analyzer, Priva, Holland).

[0128] Set point for CO2 in each phytotron was set at 420 ppmvat 940
mBar and 26° C. during the photoperiod and 450 ppmvat 940 mBar and
26° C. for the dark period.

[0129] Before being interpreted, CO2 measurements were pressure- and
temperature-compensated by the central CO2 interface & control
system, corrected for atmospheric pressure variations, temperature
variations in and outside the phytotrons and friction losses in the
guiding tubes, electric selenoids and drying filters interconnecting the
phytotrons with the central CO2 interface & control system. The
system allows for a 20 ppmvCO2 hysteresis. In order to justify air
renovation in case of excess internal CO2 concentrations,
principally during the dark period, outside air was also being evaluated
and was taken to be at least 20 ppmvbelow the internal excess
concentration for the system to activate the air exhaust system. In case
of sub set point CO2 concentrations, principally due to
photosynthesis activity during the photoperiod, pure CO2 was
injected. The amount of CO2 required was calculated and its addition
was controlled by the central CO2 interface & control system. Every
phytotron was internally equipped with a security solid state CO2
monitor (CDM4161A module, Figaro, Japan), which triggered an alarm at
CO2 levels over 1000 ppmv.

[0130] Internal air circulation. Two internal circulation ventilators
(model Master Fan Top, from Treviso Brazil) were used to ensure
circulation of 15.5 m3 of air per minute in the growing cabinets.

[0131] Air Exhaust System. An air exhaust system was used to lower the
CO2 concentration in phytotron by partially substituting the air in
the phytotron with outside air. An axial blower fan (model Taurus-H from
Ventisilva Brazil) was used to drive the exchange whereby an external
particles pre filter (model SA-3085 from Inpeca Brazil) and six activated
carbon gas-mask chemical end filters (model RC203 from Carbografite
Brazil) were used in combination to guarantee the incoming air quality.

[0132] Temperature control. York, high wall split cooling system, model
YJDA-12FS-ADA with 3.52 kW cooling capacity was used. Temperature was set
to average 26° C. (+/-1.5° C.) after air-passage through
the growing cabinets.

[0133] Humidity control. Inside each phytotron, a ventilated psychrometer
((Engineering dep. Crops Advance Brazil) was used to monitor its wet and
dry bulb temperature in a micro-fan boosted forced air stream. At a
temperature difference equal to or more than 3.0° C., the
psychrometer activated two piezo-element water nebulizers (model Polaris
Ion 35 W from Mallory of which the internal high tension "ion" generators
have been removed) until the temperature difference restores to
2.0° C. or less. This translated to vapor pressure deficit values
between 4 to 8 mBar and relative humidity values between 75% to 85%
within a temperature interval of 22° C. being close to the
evaporator unit outlet, to 27° C. at the outlet of the acryl
growing cabinets. Condensation water draining from the evaporator unit
inside each phytotron was captured for recirculation. It was passed
through a UV-C sterilization unit (Atman UV-5 W equipped with a Philips
TUV PL-S 5 W UV-C lamp) before being collected in a 40 litres collection
reservoir. The collection reservoir was equipped with a submerged pump
(Atman HF-750 18 W) to supply the interconnected water reservoirs of the
nebulizers with water in order to guarantee minimum water levels. At
minimum level, an electronic level control system activates the pump. At
full capacity, the collection reservoir drains into an external
jerry-can.

[0134] Ozone control. Ozone generators (model OP-35-3P, Grupo Interozone,
Brazil) were coupled to ozone monitors (model 202, 2B Technologies, Inc.
USA; dynamic range of 1.5 ppbv to 100 ppmv of ozone; precision 1.5 ppbv
or 2% of the reading) using a ozone monitor--generator interface
(Engineering dep. Crops Advance Brazil). At full light level (all 40
lamps activated) and no reactive surfaces other than the internal
physical phytotron structure present to interfere, the background ozone
level inside the phytotrons did not exceed 3 ppbv ozone. This was used as
the upper limit for phytotron number one. Phytotron number two was set at
30 ppbv average ozone. Phytotron number three was set at 60 ppbv average
ozone. Ozone levels of phytotron number two and three were permanently
measured at 10 second intervals and averaged per minute. Each ozone
monitor fed averaged values over an analog output to an ozone
monitor--ozone generator interface. Hysteresis was set at +/-5 ppbv.
Ozone levels were monitored close to the inlet of the two internal
circulation ventilators. The ozone generators were adapted to accept an
external digital signal from the interface.

[0135] Closed circuit video camera system. Every phytotron was equipped
with a video camera (Panasonic SDR-S7) sending its images over a TecVoz
TEC30/04LIG2 digital video record system to a server CPU, recording all
images. Over the test period the cameras have been manually put into
position to record the plants in test.

Respiration II Gas Exchange Measurements

[0136] Respiration of plant material was tested as follows: Partial oxygen
pressure drops due to oxygen consumption as result of dark respiration
were measured using phase shift fluorescence measurement. For this, a
NeoFox® Phase Measurement System was used (Fluorometer Bench version
using a QBIF600-VIS-NIR laboratory-grade bifurcated optical fiber with
FOXY-R-8 cm overcoated fluorescence oxygen needle sensor, Ocean Optics
Inc. USA).

[0137] Fluorescence phase shift was expressed as Tau in microseconds
(μs) and has an inverse and linear relation with the partial oxygen
pressure. The sensor had an atmospheric volume fraction gain of O2
per -1 μs Tau=23.79% at 26.0° C. and 936 mBar. An exemplary
graph of cotton is presented in FIG. 1

[0139] Plant material collected was always the first fully grown leaf
counting from the apical point downwards or outwards (e.g. for
monocotyls). For cotton this resulted in the third leaf under the apical
point. Per test, four leafs from four different plants were sampled.
Cotton leafs were cut in half along the mid nerve. Using a clipper, 16
leaf sections of equal surface area of 400.9 mm2 each were cut, one from
each base half and one from each top half part giving 4 sections per leaf
and 16 sections per test. The leaf sections were distributed equally over
two sub samples, one with and one without salicylhydroxamate. Using a
chirurgical blade, all sections were given 7 parallel cuts (through and
through) at approximately 2.5 mm apart over almost the full length of the
section to guarantee lateral infiltration of the test solutions and gas
exchange.

[0140] Within 10 minutes of plant sampling the sections were cut, initial
subsample weight was registered using a high precision laboratory scale
and the subsamples were placed in their solutions with and without
salicylhydroxamate for 60 minutes in the dark, to guarantee dark
adaptation.

[0141] Each sample contained 4 leaves, 4 sections per leaf providing 16
sections, 8 per subsample. In 1 subsample of 8 sections AOX was
inhibited, the other subsample served as control. Thereafter each
subsample material was tissue dried and stacked using filter paper
sections cut with the same clipper to separate individual leaf sections.
Each subsample consisted of 9 filter sections and 8 leaf sections. Each
stacked subsample was placed inside a sterile 50 ml normal tip syringe
(Embramac, Campinas Brazil) and the internal volume was set at 10 ml
bruto.

Respiration II Gas Exchange Measurements

[0142] The drop in partial oxygen vapour pressure due to oxygen
consumption as result of dark respiration was measured using phase shift
fluorescence measurement.

[0143] Both syringes (SHAM and control) were placed in a 16 litres
temperature-stabilized insulated water bath with forced water circulation
equipped with a temperature control unit (C-Control II, from Conrad
Electronic GmbH, Germany) using a high precision temperature sensor
interface set at 26.00° C. and maintaining the water temperature
within +/-0.03° C. by calculating and applying the necessary heat
injection through a 25 W glass coated resistor element (Engineering dep.
Crops Advance Brazil).

[0144] The oxygen needle probe from the NeoFox® Phase Measurement
System was inserted into the tip of the 50 mL syringe, sealed and the
complete setup was submerged in the water bath. The syringes were given
60 minutes of temperature stabilization before valid readings were taken.
The SHAM sample was read first and readings were taken every second. The
readings in general tended to linearly stabilize within 15 to 20 minutes.
Up to 20 minutes of stable trajectory was registered before switching the
syringes and the stabilizing and reading processes were repeated for the
non-SHAM subsample.

[0145] Between measurements of the SHAM and non-SHAM subsample, the probe
was allowed to stabilize. The readings were collected into computer
memory. From this, the Total Dark Respiration (TDR) including the AOX
(non-SHAM) and Total Dark Respiration (TDR) excluding the AOX (SHAM) were
determined. The results for cotton are provided in Table 1.

[0146] In addition, full leaf Total Dark Respiration (TDR) measurements
without the use of any pre-treatment solution (no SHAM and pretreatment
control) were executed for cotton, corn and rice. Data (tables 2, 3 and
4) show clear positive correlation between a) total respiration per unit
of fresh weight and ozone level, and b) total respiration per unit of dry
weight and ozone level.

[0147] The plants that passed their ontogenic phase in the 0 ppbv ozone
ambient have an AOX respiration rate of 26.0% of the total 100% Total
Dark Respiration rate. When placed in the 30 ppbv ozone ambient, the AOX
respiration increased to 30.3%, +16.5% compared to the initial AOX
respiration rate and those placed in the 60 ppbv ozone ambient increased
their AOX respiration to 39.6%, +52.5% compared to the initial AOX
respiration rate.

[0148] When returned to the 0 ppbv ozone ambient in which the plants lived
their ontogenic phase, the AOX respiration of the plants which had passed
time in the 60 ppbv ozone ambient returned fully to its initial level.

[0149] Plants grown up in the 60 ppbv ozone level ambients did not lower
their AOX respiration during their stay in the zero ozone level ambient
and maintained their high AOX respiration rate.

[0150] The AOX respiration rate expressed as percentage of the total dark
respiration is defined as the base level during the ontogenic phase.

[0151] Plants with AOX levels defined during the ontogenic phase increase
their AOX respiration when exposed to oxidative stress induced by higher
ozone levels, but when the oxidative stress is reduced, cotton showed its
AOX respiration rate to fully return to base level within 3 days. When
exposed to ozone levels lower than the base level of the plants, the AOX
respiration does not go below the base level defined during the ontogenic
phase.

[0152] These findings have great implications for methods for preventing
inefficient respiration in plants, and hence for increasing plant yield.